Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae

Selenium Attenuates Chronic Heat Stress-Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in Mouse Granulosa Cells

Heat stress induces apoptosis in various cells. Selenium, an essential micronutrient, has beneficial effects in maintaining the cellular physiological functions. However, its potential protective action against chronic heat stress (CHS)-induced apoptosis in granulosa cells and the related molecular mechanisms are not fully elucidated. In this study, we investigated the roles of selenium in CHS-induced apoptosis in mouse granulosa cells and explored its underlying mechanism. The heat treatment for 6–48 h induced apoptosis, potentiated caspase 3 activity, increased the expression levels of apoptosis-related gene BAX and ER stress markers, glucose-regulated protein 78 (GRP78), and CCAAT/enhancer binding protein homologous protein (CHOP) in mouse granulosa cells. The treatment with ER stress inhibitor 4-PBA significantly attenuated the adverse effects caused by CHS. Selenium treatment significantly attenuated the CHS- or thapsigargin (Tg, an ER stress activator)-induced apoptosis, potentiation of caspase 3 activity, and the increased protein expression levels of BAX, GRP78, and CHOP. Additionally, treatment of the cells with 5 ng/mL selenium significantly ameliorated the levels of estradiol, which were decreased in response to heat exposure. Consistently, administering selenium supplement alleviated the hyperthermia-caused reduction in the serum estradiol levels in vivo. Together, our findings indicate that selenium has protective effects on CHS-induced apoptosis via inhibition of the ER stress pathway. The current study provides new insights in understanding the role of selenium during the process of heat-induced cell apoptosis

Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored

Proteomic Identification of Differentially Expressed Proteins between Male and Female Plants in <i>Pistacia chinensis</i>

<div><p><i>Pistacia chinensis</i> is a strict dioecious plant with male and female flowers in individuals. In China, <i>P. chinensis</i> is widely planted for biodiesel oil due to high oil content in seeds. In practice it requires to grow more female plants for biodiesel production. At present, there are still no reliable methods for sex determination during the long juvenile stage of this species. In order to develop protein molecular markers for sex determination in <i>P. chinensis</i>, proteomic approach was used to identify differentially expressed proteins between male and female plants. Vegetative organs (leaf and stem) rather than reproductive organs/tissues were used for protein extraction so as to develop protein markers which can be used in siblings before flowering. Protein was extracted using a phenol-based protocol. By using two-dimensional electrophoresis, a total of 10 protein spots were found to be differentially expressed in leaf and stem between both sexes, of which 7 were successfully identified by mass spectrometry and matched to 6 functional proteins such as NB-ARC domain containing protein, light harvesting chlorophyll a/b binding protein, asorbate peroxidase (APX), eukaryotic translation initiation factor 5A2, temperature-induced lipocalin (TIL) and phosphoglycerate kinase (PGK). The sex-related difference displayed in a tissue-specific way, especially in stem. PGK existed in high abundance in stem phloem in the female, but was almost not detected in the male; APX and two TIL species were highly abundant in the stem of male plants, while their abundance was much lower in female plants. Moreover, these abundance differences were further confirmed in individual plants. Hence, it is assumed that APX, PGK and TIL might be promising candidates to serve as protein molecular markers for sex determination in <i>P. chinensis</i>. Our results form the basis for a further understanding of the biochemical mechanisms of sex determination in <i>P. chinensis</i>.</p></div

2-DE identification of differentially expressed proteins in stem xylems between male and female plants (10-year-old) in <i>Pistacia chinensis</i>.

<p>Stem was sampled in winter. A mixed tissue powder from three different male or female individuals was used for protein extraction. <b>A</b>, 2-DE profile of xylem proteins from female plants as reference. <b>B</b>, magnified gel regions containing spots X1-X3, accompanied by column configuration of relative abundance (generated by PDQUEST). Spot X2 failed to be identified by MS/MS. f = female; m = male.</p

2-DE identification of differentially expressed proteins in stem xylem between male and female individuals (10-year-old) in <i>Pistacia chinensis</i>.

<p><i>Up panel</i>, two representative CBB-stained gels, xylem was sampled in winter; <i>down panel</i>, magnified gel regions containing spots X1 (APX) out of a total of 8 individuals, of which 1–4 were sampled in winter and 5–8 sampled in autumn. f = female; m = male.</p

2-DE identification of differentially expressed proteins in leaves between male and female plants (10-year-old) in <i>Pistacia chinensis</i>.

<p>A mixed tissue powder from three different male or female individuals was used for protein extraction. <b>A</b>, 2-DE profile of leaf proteins from female plants as reference. <b>B</b>, magnified gel regions containing spots L1 and L2, accompanied by column configuration of relative abundance (generated by PDQUEST). f = female; m = male. OEE2 = oxygen-evolving enhancer protein 2. Arrow indicates the prominent spot Rubisco.</p

2-DE identification of differentially expressed proteins in stem phloem between male and female individuals (10-year-old) in <i>Pistacia chinensis</i>.

<p>Two representative 2-DE gels from three biological replicas are shown. Stem was sampled in winter. f = female; m = male.</p

Differentially expressed proteins identified by MS/MS analysis in vegetative tissues between male and female plants in <i>Pistacia chinensis</i>.

<p><b>Note:</b> -, undetected by PDQUEST analysis. Spot P3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064276#pone-0064276-g003" target="_blank">Figure 3</a> and spot X2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064276#pone-0064276-g004" target="_blank">Figure 4</a> failed to be identified by MS/MS and are not listed in the table.</p>a<p>, subcellular l in UniProtKB;</p>b<p>, subcellular localization predicted by software.</p><p>PM = Plasma membrane.</p

Workflow for sequential protein extraction protocol.

<p>Acetone tissue powder of <i>Pinellia ternata</i> tubers was sequentially extracted by 10% acetic acid, followed by SDS-containing buffer.</p